Organisms such as microorganisms and viruses can play a significant role in the lives of plants, animals and humans for a wide variety of reasons. For example, bacterial microorganisms are involved in the spoilage of meat, wines, vegetables and dairy products and can render these foods unpalatable or even dangerous, leading to food poisoning such as that caused by Staphylococcus aureus or Clostridium botulinum. Likewise, bacteria can be of significant importance in various industries due to their fermentation capabilities. Especially significant is the fact that about 200 species of bacteria are pathogenic for humans. For example, invasive bacteria are responsible for human diseases such as several forms of pneumonia, diphtheria, leprosy, plague, dysentery, tuberculosis, cholera, lockjaw, tetanus, syphilis, gonorrhea, typhoid fever, and the like.
Similarly, viruses play a major role in infectious diseases. For example, among the many diseases caused by viruses in humans, there may be mentioned, for example, the common cold, rabies, poliomyelitis, yellow fever, encephalitis, hemorrhagic fevers, influenza and other respiratory diseases, hepatitis, warts, chicken pox, shingles, acute diarrhea, fever blisters (herpes simplex), mumps, measles, rubella, acquired immune deficiency syndrome (AIDS), certain cancers, Creutzfeldt-Jakob disease, infant gastroenteritis and the like. Viruses can also cause a wide variety of diseases in plants, such as for example, those cause by turnip yellow mosaic virus, tobacco mosaic virus, potato X virus and the like.
Likewise, fungi can be a significant source of health problem in humans or plants. For example, Madurella mycetoni fungi can produce maduromycosis or other foot infections and many fungal forms of mushrooms are known for their deadly consequences.
It is therefore clear that it is of major importance to be able to detect the presence of organism such as microorganism, for example, such as bacterium or fungi, or virus in various environments for both their desired and undesired consequences.
Over the years, a wide variety of detection techniques have been utilized for the detection of the presence of microorganism or virus in samples including incubation and the like. Polymerase chain reaction (PCR) is a recently developed significant and powerful technique for polynucleotide amplification. The technique is disclosed, for example, in U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159 and 4,965,188. PCR may be generally described as follows. The technique is an enzymatic, in vitro synthesis method for replicating or amplifying specific target polynucleotide sequences in samples. The technique employs polymerase, deoxynucleoside triphosphates and two oligonucleotide primers that hybridize to opposite strands of the polynucleotide sample and flank the region of interest in the target polynucleotide sequence. Experimental amplification of the target sequence is obtained by a repetitive series of steps comprising template denaturation, primer annealing and extension of the annealed primers by polymerase, generally referred to as thermal cycling steps. Such a PCR technique is capable of producing amplification of the target sequence by a factor of up to about 10.sup.9 which can then be subject to an assay procedure appropriate for the target sequence. However, even PCR is time consuming due to the time required, about 2 hours, for the repetitive cycling steps required for amplification and replication.
It is therefore highly desirable that a method be provided for easy and rapid analysis of samples for the determination of the possible presence of organisms such as microorganisms or viruses. It is even more desirable that such a method be provided that will enable detection of even very low levels of organism, e.g. levels of 1000 or less organisms per sample, more preferably as low as about 100 or less, and even more preferably detection of as low as a single organism per sample. A further object is to provide such an assay method that can be employed without having to amplify or multiply any components of the organism and therefore can be conducted quickly and in numerous locations. Yet another object of this invention is to provide a quick and easy assay procedure that is simply to perform and not requiring complex equipment and thus suitable for on-site field use.